The identification of rare antigen-specific T cell populations in peripheral blood or tissues following vaccination can be achieved either by increasing the sensitivity of an assay or by reducing the denominator of the measured cell population in which the antigen-specific cells are located. A strategy that reduces this denominator can be achieved by using flow cytometry and choosing multiple surface or intracellular markers to accurately define the T cell subsets of interest. In addition, the definition of effector function of antigen-specific T cells will likely require multiple intracellular and surface markers. Thus, we believe that multiparameter flow cytometry (MFC), which may employ up to 11 phenotypic or functional markers, will be a useful tool for the study of the quantity and function of T cells induced after vaccination. The overall goal of this project is to bring together a working group of investigators to help to develop the tools, reagents and expertise needed to make the use of MFC a reliable and practical approach that can be used in HIV vaccine research. The field has a number of important roadblocks that will be addressed. These include the basic problem that there is no standard hardware configuration of lasers, filters and detectors (PMTs or photomultiplier tubes), limited commercial software for collecting and analyzing the data, and no agreement on either the surface markers, intracellular markers, fluorophores, or combinations of stains that should be used for future assessment of antigen-specific T cell populations. Lastly, the techniques developed to date have not been rigorously applied to vaccine studies, and thus the performance characteristics in settings in which the memory population may be small is unknown. The specific AIMS of this proposal are: 1) To develop a T cell anchored system of phenotyping for the analysis of rare antigen-specific CD4+ and CD8+ populations, and to define the performance characteristics of this system. The variability between individuals, and the inter-assay variability will be determined using both fresh and cryopreserved lymphocytes. 2) To conduct a multicenter trial of our validated panel by sending specimens from chosen vaccinated volunteers to four centers with multiparameter flow capability. We will also determine whether the samples should be stimulated on site, fixed and mailed, or whether they can be mailed, and then stimulated on site. 3) To apply this methodology to determine the CD4 and CD8 T cell responses to vaccination, with a special emphasis given to the Th1/Th2 phenotype of the responding cells. We have begun a series of adjuvant trials using hepatitis B vaccine and gp120. The panel developed in each of the AIMS above will be applied to these studies to determine whether the differences previously seen in antibody outcomes can be determined at the T cell phenotype and antigen-specific level.